The effectiveness of KEL and RHCE fetal genotype assessment in alloimmunized women by minisequencing

Česká verzia

Authors: V. Durdová ¹ ; J. Böhmová ²; T. Kratochvílová ¹ ; R. Vodička ²; I. Holusková ³; K. Langová ⁴ ; M. Lubušký ¹
Authors place of work: Porodnicko-gynekologická klinika LF UP a FN, Olomouc, přednosta prof. MUDr. R. Pilka, Ph. D. ¹; Ústav lékařské genetiky LF UP a FN, Olomouc, přednosta prof. MUDr. M. Procházka, Ph. D. ²; Transfuzní oddělení FN, Olomouc, vedoucí oddělení MUDr. D. Galuszková, Ph. D., MBA ³; Ústav lékařské biofyziky LF UP, Olomouc, přednostka prof. RNDr. H. Kolářová, CSc. ⁴
Published in the journal: Ceska Gynekol 2020; 85(3): 164-173
Category: Original Article

Summary

Objective: To evaluate the effectiveness of the fetal KEL and RHCE genotype assessment in alloimmunized pregnant women by minisequencing.

Design: Prospective cohort study.

Setting: Obstetrics and Gynecology Clinic of the Faculty of Medicine UP and the University Hospital Olomouc; Institute of Medical Genetics of the Faculty of Medicine UP and the University Hospital Olomouc; Transfusion Department of the University Hospital Olomouc; Institute of Biophysics of the Faculty of Medicine UP Olomouc.

Subject and method: In the years 2001–2019, 366 samples of pregnant women in the first and second trimester were assessed KEL (n = 327) or RHCE (n = 39) genotype from the free fetal DNA circulating in the peripheral blood by minisequencing. The genotype of the fetus was verified from the buccal smear of the newborn.

Results: The KEL genotype was assessed in 327 women (the presence of a variant of the KEL1 alele, which corresponds to the presence of the erythrocyte antigen “K“. The analysis failed in 2 cases (2/327), 16 heterozygote women (KEL1/KEL2) were excluded and in the case of 309 homozygote women (KEL2/KEL2) the fetal KEL genotype was assessed.

In the case of 95.8% of the fetuses (296/309) and 95.5% of the newborns (295/309), the KEL2/KEL2 genotype was assessed. In the case of 4.2 % of the fetuses (13/309) and 4.5% of the newborns (14/309), the KEL1/KEL2 genotype was assessed. The sensitivity was 92.86%. The specificity was 100%.

The RHCE genotype was assessed in 39 women. In the case of 22 women, the presence of a variant of the RHCE gene, which corresponds to the presence of the erythrocyte antigen “C“/“c“, was assessed. 5 heterozygote women (C/c) were excluded. In the case of 11 homozygote women (C/C), the RHCE genotype was assessed. In the case of 64% (7/11) of the fetuses and newborns, the C/c genotype was assessed, in the case of 36% (4/11) the C/C genotype was assessed.

In the case of 6 homozygote women (c/c), the RHCE genotype was assessed.

In the case of 67% (4/6) of the fetuses and newborns, the C/c genotype was assessed, in the case of 33% (2/6) the c/c genotype was assessed. The sensitivity and specificity were 100%.

In the case of 17 women, the presence of the variant of the RHCE gene, which corresponds to the presence of the erythrocyte antigen “E“/“e“, was assessed. 1 heterozygote woman (E/e) was excluded. In the case of 16 homozygote women (e/e), the RHCE genotype was assessed. In the case of 75% (12/16) of the fetuses and newborns, the e/e genotype was assessed, in the case of 25% (4/16) the E/e genotype was assessed. The sensitivity and specificity were 100%.

Conclusion: The minisequencing method using the capillary electrophoresis enabled a reliable detection of the fetal KEL and RHCE genotype from the peripheral blood of pregnant women.

Keywords:

pregnancy – alloimmunization – cell free DNA – KEL and RHCE genotype

Zdroje

1. Basu, S., Kaur, R., Kaur, G. Hemolytic disease of the fetus and newborn: Current trends and perspectives. Asian J Transfusion Sci, 2011, 5, p. 3–7.

2. Bohmova, J., Vodicka, R., Lubusky, M., et al. Clinical potential of effective noninvasive exclusion of KEL1-positive fetuses in KEL1-negative pregnant women. Fetal Diag Ther, 2016, 40, p. 48–53.

3. Bowman, JM., Harman, CR., Manning, FA., Pollock, JM. Erythroblastosis fetalis produced by anti-k. Vox Sang,1989, 56, 3, p. 187–189, Erratum in: Vo. Sang, 1990, 58, 2, p. 139.

4. British Committee for Standards in Haematology. Milkins, C., Berryman, J., et al. Guidelines for pre-transfusion compatibility procedures in blood transfusion laboratories. British Committee for Standards in Haematology. Transfus Med, 2013, 23, p. 3–35.

5. Daniels, G., Bromilow, I. Essential Guide to Blood Groups. 1th ed. Oxford: Blackwell Publishing, 2007.

6. Daniels, G., Green, C. Expression of red cell surface antigens during erythropoesis. Vox Sang, 2000, 78 (Suppl. 2), p. 149–153.

7. Daniels, G., Hadley, A., Green, C. Fetal anemia due to anti- -K may result from immune destruction of early erythroid progenitors. Transfus Med, 1999, 9 (suppl. 1), p. 16 (abstract).

8. Durdová, V., Holusková, I., Kratochvílová, T., et al. Klinický význam neinvazívního stanovení KEL genotypu plodu v managementu těhotenství s rizikem rozvoje hemolytické nemoci plodu a novorozence. Postgrad Med, 2016, 18, s. 358–361.

9. Hackney, DN., Knudtson, EJ., Rossi, KQ., et al. Management of pregnancies complicated by anti-c isoimmunization. Obstet Gynecol, 2004, 103, p. 24–30.

10. Holuskova, I., Lubusky, M., Studnickova, M., et al. Erytrocytární aloimunizace u těhotných žen, klinický význam a laboratorní diagnostika. Ces Gynek, 2013, 78, s. 89–99.

11. Holuskova, I., Lubusky, M., Studnickova, M., et al. Incidence of erythrocyte alloimmunization in pregnant women in olomouc region. Ces Gynek, 2013, 78, s. 56–61.

12. Hromadnikova, I., Vechetova, L., Vesela, K., et al. Noninvasive fetal RHD and RHCE genotyping using real-time PCR testing of maternal plasma in Rh-negative pregnancies. J Histochemistry Cytochemistry, 2005, 53, p. 1–5.

13. Kratochvilova, T., Durdova, V., Strasilova, P., et al. Klinický význam neinvazívního stanovení RHD a RHCE genotypu plodu v managementu těhotenství s rizikem rozvoje hemolytické nemoci plodu a novorozence. Postgrad Med, 2016, 18, s. 362–369.

14. Ľubušký, M., Procházka, M., Hálek, J., Klásková, E. Management těhotenství s rizikem rozvoje hemolytické nemoci plodu a novorozence. Postgrad Med, 2016, 18, s. 352–357.

15. Masopust, J., Písačka, M. Praktická imunohematologie. Erytrocyty. Praha: Mladá fronta, 2016, 1. vyd.

16. McKenna, DS., Nagaraja, HN., O’Shaughnessy, RW. Management of pregnancies complicated by anti-Kell isoimmunization. Obstet Gynecol, 1999, 93, p. 667–673.

17. Moinuddin, I., Fletcher, C., Millward, P. Prevalence and specificity of clinically significant red cell alloantibodies in pregnant women – a study from a tertiary care hospital in Southeast Michigan. Journal of blood medicine 2019, 10, p. 283–289

18. Pal, M., Williams, B. Prevalence of maternal red cell alloimmunisation: a population study from Queensland, Australia. Pathology, 2015, 47, p. 151–155.

19. Reid, M., Lomas-Francis, C. The blood group antigen facts. 2nd ed, New York: Elsevier Academic Press, 2004.

20. Smith, HM., Shirey, RS., Thoman, SK., et al. Prevalence of clinically significant red blood cell alloantibodies in pregnant women at a large tertiary-care facility. Immunohematology, 2013, 29, p. 127–130.

21. van Wamelen, DJ., Klumper, FJ., de Hass, M., et al. Obstetric history and antibody titer in estimating severity of Kell alloimmunization in pregnancy. Obstet Gynecol, 2007, 109, p. 1093–1098.

22. Wenk, RE., Goldstein, P., Felix, JK. Alloimmunization by hr‘(c), hemolytic disease of newborns, and perinatal management. Obstet Gynecol, 1986, 67, p. 623–626.